Smad7在肠道发育与肿瘤形成中的分子机制

High expression of Smad7 is associated with tumorigenesis, progression, and prognosis of intestinal tumor. We participated in the engineering of Rosa-Smad7 conditional knock-in (CKI) mice. By cross-breeding these mice to the mice with specific expression of Cre in intestinal epithelia and mesenchyme respectively using Cre-LoxP system, we found some of offspring may die in embryonic period while others survived postnatally, but show enhanced intestinal tumorogenesis, suggesting that Smad7 affects the intestinal development and homeostasis. To explore the role of Smad7 in intestinal development and tumorigenesis in different growing ages, this project proposes to up-regulate Smad7 expression in intestinal epithelial, crypt stem cell and/or mesenchymal at different development stage selectively, then characters the malformation and tumorigenesis of intestine. We will also up/down regulate the effector molecules of Smad7 in epithelia, mesenchyme and muscle cells, to further confirm the role of Smad7 played in intestine development and function maintaining especially the interaction between epithelia and mesenchyme, using RNA-Seq, IHE/IF, qPCR, Western Blotting and other techniques. The proposed studies will help unlock the molecular mechanism of Smad7 in the intestinal development and tumorigenesis.

Smad7的高表达与肠道肿瘤的发生、发展和预后相关。申请人参与了Rosa-Smad7条件性基因敲入(CKI)小鼠的构建，用Cre-LoxP系统在小鼠肠道上皮或间质稳定表达Smad7，该CKI小鼠或在胚胎时期死亡，或在出生后存活，但自发产生不同类型的肠道肿瘤，提示Smad7影响胚胎期和出生后肠道细胞的发育及功能维持。为进一步阐明Smad7在不同鼠龄肠道发育及肿瘤形成中的作用机理，本课题在肠上皮、隐窝成体干细胞及间质分时段选择性上调Smad7，检测该基因对胎鼠、幼鼠及成年鼠肠道发育的影响。结合体外细胞过表达/剔降实验，用RNA-Seq、IHC/IF、qPCR、Western Blottting等方法鉴定Smad7影响肠上皮细胞、间质细胞、肌细胞发育与分化的效应分子，进一步证实Smad7在肠道形成和生理稳态，特别是上皮与间质交互作用中的功能，有望明确Smad7在肠道发育及肿瘤形成中的分子机制。

Smad7作为转化生长因子（TGF）-β的抑制剂在人结肠癌组织和结直肠癌细胞中的表达较高，我们制作了RSmad7条件性敲入（CKI）自发性结直肠肿瘤动物模型，结果发现CKI小鼠自发形成的癌组织中β-catenin高度核阳性，同时Wnt信号通路的靶分子CD44、Sox9等也被激活。为了进一步验证Smad7与Wnt信号通路在结肠肿瘤形成中的相互作用，我们制作了上皮组织中敲入Smad7且半敲除APC的小鼠模型，发现Smad7能加速小鼠肠道肿瘤的形成和恶化。此外，在结直肠癌细胞系中上调Smad7的表达能够促进结直肠癌细胞的生长、迁移和克隆形成，减少细胞凋亡。下调Smad7的表达则抑制结直肠癌细胞的生长、迁移及克隆形成，凋亡细胞数增加。我们还发现在结直肠癌细胞中过表达Smad7能够降低β-catenin的磷酸化水平，促使β-catenin积聚并向核内移位，Wnt/β-catenin信号通路中的APC/AXIN/GSK3β复合体的表达量降低，WNT信号通路的下游靶基因MMP7、c-Myc、CyclinD1等表达增高。综上所述，我们的研究结果验证了Smad7在结直肠癌发生和发展中的作用，提示了Smad7可能通过介导Wnt/β-catenin信号通路促进结直肠癌的进展，为结直肠癌的治疗及预后提供了理论基础。